Colorectal adenoma subtypes exhibit signature molecular profiles: unique insights into the microenvironment of advanced precancerous lesions for early detection applications

Academic Edi o s: Lisa do Bosca and
Se ge Roche
Recei ed: 17 Decembe 2024
Re ised: 24 Janua y 2025
Accep ed: 8 Feb ua y 2025
Published: 14 Feb ua y 2025
Ci a ion: Mancuso, F.M.;
Higa eda-Alma az, J.C.;
Canal-Nogue , P.; Be ossi, A.;
Pe e a-Lluna, A.; Roeh l, M.H.A.;
K uusmaa, K. Colo ec al Adenoma
Sub ypes Exhibi Signa u e Molecula
P o iles: Unique Insigh s in o he
Mic oen i onmen o Ad anced
P ecance ous Lesions o Ea ly
De ec ion Applica ions. Cance s 2025,
17, 654. h ps://doi.o g/10.3390/
cance s17040654
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
Colo ec al Adenoma Sub ypes Exhibi Signa u e Molecula
P o iles: Unique Insigh s in o he Mic oen i onmen o
Ad anced P ecance ous Lesions o Ea ly De ec ion Applica ions
F ancesco Ma ia Mancuso 1, Juan Ca los Higa eda-Alma az 1, Pol Canal-Nogue 1, A ianna Be ossi 2,
Alexand e Pe e a-Lluna 3,4 , Michael He be Alexande Roeh l 5and K is i K uusmaa 2,*
1Uni e sal Diagnos ics S.A., 41013 Se ille, Spain; [email p o ec ed] (F.M.M.);
[email p o ec ed] (J.C.H.-A.); [email p o ec ed] (P.C.-N.)
2Resea ch & De elopmen , Uni e sal Diagnos ics d.o.o., 1000 Ljubljana, Slo enia;
[email p o ec ed]
3B2SLab, Ins i u e o Resea ch and Inno a ion in Heal h (IRIS), Uni e si a Poli ècnica de
Ca alunya—Ba celonaTech, 08028 Ba celona, Spain; alexand e.pe [email p o ec ed]
4Ne wo king Biomedical Resea ch Cen e in he Subjec A ea o Bioenginee ing, Bioma e ials and
Nanomedicine (CIBER-BBN), 28029 Mad id, Spain
5Depa men o Pa hology, Be h Is ael Deaconess Medical Cen e , Ha a d Medical School,
Bos on, MA 02215, USA; [email p o ec ed]d.edu
*Co espondence: k is i.k [email p o ec ed]
Simple Summa y: Colo ec al cance o igina es om benign g ow hs in he colon known
as adenomas. Unde s anding how hese adenomas become malignan is c ucial o he
ea ly de ec ion and p e en ion o colo ec al cance . This s udy examines DNA me hyla ion
and gene ic di e ences among a ious ypes o ad anced adenomas o iden i y po en ial
ma ke s ha indica e p og ession owa d cance . By analyzing DNA me hyla ion pa e ns,
gene copy-numbe changes, and mu a ions in hese lesions, pa icula signals and pa hways
associa ed wi h each sub ype we e ound. These disco e ies can help e ine sc eening
me hods o de ec dis inc adenomas lesions a an ea ly s age. By p o iding new insigh s
in o he ini ial changes leading o colo ec al cance , hese indings may signi ican ly impac
app oaches o p e en ion and ea ly in e en ion s a egies.
Abs ac : Backg ound: Colo ec al cance (CRC) is cha ac e ized by he uncon olled g ow h
o malignan colonic o ec al c yp epi helium. Abou 85% o CRCs e ol e h ough a
s epwise p og ession om ad anced p ecance ous adenoma lesions. A be e unde s and-
ing o he e olu ion om adenoma o ca cinoma can p o ide a window o oppo uni y
no only o ea ly de ec ion and he apeu ic in e en ion bu po en ially also o cance
p e en ion s a egies. Me hods: This s udy in es iga es he he e ogeneous me hyla ion,
copy-numbe al e a ion (CNA), and mu a ion signals o his ological adenoma sub ypes
in he con ex o p og ession om no mal colon o ad anced p ecance ous lesions (APLs)
and ea ly-s age CRC. Resul s: Di e en ial me hyla ion analysis e ealed 2321 signi ican ly
al e ed egions among APLs: 137 hype me hyla ed egions in se a ed s. ubula , 2093
in se a ed s. ubulo illous, and 91 in ubula s. ubulo illous adenoma sub ypes. The
mos di e en ia ing pa hways o se a ed adenomas belonged o cAMP signaling and he
egula ion o plu ipo ency o s em cells, while egions sepa a ing ubula and ubulo illous
sub ypes we e en iched o WNT signaling. CNA e en s we e mos ly p esen in ubula
o ubulo illous adenomas, wi h he mos equen signals being seen in ch omosomes
7, 12, 19, and 20. In con as , ea ly-s age CRC exhibi ed signals in ch omosomes 7, 8, and
20, indica ing di e en p ocesses be ween APL and ea ly-s age CRC. Mu a ions ein o ce
sub ype-le el di e ences, showing speci ic al e a ions in each sub ype. Conclusions: These
Cance s 2025,17, 654 h ps://doi.o g/10.3390/cance s17040654
Cance s 2025,17, 654 2 o 19
indings a e especially impo an o de eloping ea ly de ec ion o cance p e en ion es s
ying o cap u e adenoma signa u es.
Keywo ds: colo ec al cance ; adenoma; ad anced p ecance ous lesion; me hyla ion; copy-
numbe al e a ions; pa hway en ichmen
1. In oduc ion
Colo ec al cance (CRC) is a he e ogeneous malignancy o igina ing om he epi helial
cells lining he colon and ec um, posing a signi ican global heal h bu den wi h o e
1.9 million new cases
and 935,000 dea hs in 2020 [
1
]. I is he hi d mos diagnosed cance
and he second leading cause o cance - ela ed mo ali y wo ldwide. Despi e ad ancemen s
in sc eening and ea men , CRC con inues o exhibi high mo ali y a es, emphasizing
he need o deepe molecula insigh s o enhance ea ly de ec ion and de elop a ge ed
he apeu ic in e en ions [2].
CRC a ises h ough a complex in e play o gene ic and en i onmen al ac o s. While
he edi a y synd omes such as amilial adenoma ous polyposis (FAP) and Lynch synd ome
con ibu e o a subse o cases [
3
], mos CRCs occu spo adically and a e in luenced by
li es yle ac o s like die , physical inac i i y, and smoking. Howe e , in many ins ances, he
exac causes emain unknown, highligh ing he need o u he esea ch in o addi ional
con ibu ing ac o s [
4
,
5
]. The adenoma–ca cinoma sequence, i s p oposed by Vogels ein,
ou lines he s epwise p og ession o CRC, wi h gene ic al e a ions in key oncogenes and
umo supp esso s such as APC [
6
], KRAS [
7
], and TP53 [
8
], playing pi o al oles in umo
ini ia ion and p og ession. Howe e , g owing e idence unde sco es he ole o epigene ic
modi ica ions, pa icula ly DNA me hyla ion changes, in CRC pa hogenesis [9,10].
Abe an DNA me hyla ion has been iden i ied as a majo d i e o CRC de elopmen
by silencing umo supp esso genes and ac i a ing oncogenic pa hways [
11
]. These
abe an me hyla ed egions a e now ecognized as po en ial diagnos ic and p ognos ic
bioma ke s [
12
], o e ing insigh s in o disease he e ogenei y and ea men esponse [
13
].
Recen s udies ha e e ealed ha CRC umo s ha bo hund eds o hype me hyla ed
egions compa ed wi h adjacen no mal mucosa, highligh ing he impo ance o epigene ic
dys egula ion in colo ec al ca cinogenesis [14–16].
Ad anced p ecance ous lesions (APLs), including ubula adenomas (TAs), il-
lous/ ubulo illous adenomas (VAs/TVAs), and se a ed adenomas (SAs), exhibi dis inc
gene ic and epigene ic p o iles ha con ibu e o hei a iable isk o malignan ans o ma-
ion [
17
]. Se a ed adenomas, whose p og ession is an excep ion o he adenoma–ca cinoma
sequence [
18
], a e equen ly associa ed wi h he CpG island me hyla o pheno ype (CIMP)
and mic osa elli e ins abili y (MSI), u he unde sco ing he impo ance o epigene ic
al e a ions in CRC p og ession [19].
Al hough his opa hology emains a co ne s one in diagnosing and sub yping APLs,
accu a ely cha ac e izing hese lesions can be challenging. S udies epo a iabili y in
assessmen s among pa hologis s, esul ing in inconsis encies ha may in luence pa ien
managemen and ou comes [
20
]. In ce ain cases, such in e p e i e di e ences can lead o
unce ain ies ega ding lesion ma gins, po en ially hinde ing imely decision making [
21
].
Gi en he complexi y and he e ogenei y o APLs, supplemen ing his opa hological e alu-
a ion wi h addi ional molecula o mul i-omics app oaches can help enhance diagnos ic
accu acy and imp o e pa ien ca e.
Despi e hese challenges, no diagnos ic ools ha e been speci ically de eloped o
alida ed o APLs, u he unde sco ing he c i ical need o imp o ed me hodologies o
Cance s 2025,17, 654 3 o 19
ensu e accu a e diagnosis and e ec i e clinical decision making. Cu en ly, mos a ailable
ools a e designed o b oade CRC applica ions, lacking speci ici y o APLs. A mul i-
omics app oach, which in eg a es mul iple laye s o biological in o ma ion, is essen ial
o achie ing a comp ehensi e and sys emic unde s anding o he molecula landscape in
CRC [
22
]. Such in eg a i e analyses no only imp o e diagnos ic accu acy bu also acili a e
he iden i ica ion o no el, mo e eliable bioma ke s and he apeu ic a ge s, enabling
pe sonalized and mo e e ec i e pa ien managemen [
23
]. Recen mul i-omics s udies
ha e al eady p o ided c i ical insigh s in o global DNA me hyla ion pa e ns [
24
], gene
exp ession, CNVs, and mu a ion analysis [
25
], as well as me abolomics [
26
], highligh ing
he po en ial o hese ad anced app oaches o e olu ionize he diagnosis and ea men o
CRC. Howe e , he applica ion o hese mul i-omics s a egies emains la gely unexplo ed
in he con ex o APLs, u he emphasizing he need o dedica ed esea ch e o s o
de elop APL-speci ic diagnos ic ools.
This s udy aims o iden i y key molecula ea u es encompassing DMRs, CNVs, and
genomic mu a ions ac oss sub ypes o APLs by using a mul i-omics app oach ha in eg a es
genome-wide da a. By explo ing hese laye s o biological in o ma ion, we aim o e ine
APL classi ica ion and po en ially imp o e cu en isk s a i ica ion models. Ou indings
may con ibu e o mo e in o med decision making, ea lie de ec ion, and be e - a ge ed
he apeu ic s a egies in he managemen o colo ec al cance .
2. Ma e ials and Me hods
2.1. Samples
In his mul i-cen e e ospec i e s udy, a o al o 168 esh ozen (FF) issue sam-
ples we e ob ained om di e en in e na ional biobank eposi o ies, including TCBN
(Caen, F ance), VCB (Melbou ne, VIC, Aus alia), Indi umed (Hambu g, Ge many),
IDIBAPS (Ba celona, Spain), LBIH Biobank (Li e pool, UK), and Dx Biosamples (San
Diego, CA, USA).
A o al o 96 and 46 pai ed umo issues and no mal adjacen issues om 48 APLs
and 23 s age I CRCs we e designedly selec ed o omics de ec ion. An independen se o
APL samples we e used o alida ion pu poses.
Tissue samples wi h di e en his ological backg ounds (sessile se a ed lesions (SSLs),
ubula adenomas (TAs), o illous/ ubulo illous adenomas (VAs/TVAs)) we e used o
ob ain DNA o whole-genome bisul i e sequencing.
Fo ou analysis, we de ine wo samples as pai ed when ex ac ed om he same
pa ien hough o di e ing issue o sample ype.
Demog aphic sample dono ea u es a e lis ed in Table 1and Supplemen a y Table S1.
Table 1. Rele an cha ac e is ics o pa ien s in his s udy.
APL APL
(Valida ion) CRC S age I
n 48 26 23
Age median (IQR), yea s
( ange)
68.5 (14.75)
(39–86)
68.5 (19.75)
(23–84)
68 (13)
(37–81)
Gende emale/male, n 17/31 16/10 14/9
His ology
SSL, n 7 7
TA, n 22 4
VA/TVA, n 19 15
Cance s 2025,17, 654 4 o 19
2.2. Sample P epa a ion and Whole-Genome Bisul i e Sequencing (WGBS)
Genomic DNA (gDNA) om FF issue was ex ac ed by using a DNeasy Blood &
Tissue ki (Qiagen, Valencia, CA, USA) acco ding o he p o ocol by he manu ac u e .
Ex ac ed gDNA was hen agmen ed in o segmen s o abou 400 bp wi h a Co a is
S220 ul asonica o . The ex ac ed and sh edded gDNA was bisul i e-con e ed wi h he
EZ DNA Me hyla ion-Ligh ning ki (Zymo Resea ch, Inc., I ine, CA, USA). Sequenc-
ing lib a ies we e p epa ed om he bisul i e-con e ed DNA by using he Accel-NGS
Me hyl-seq DNA lib a y ki (Swi Biosciences (Ann A bo , MI, USA) and consequen ly
sequenced o an a e age dep h o 22.5x wi h No aSeq6000 (Illumina, San Diego, CA, USA)
equipmen , using pai ed-end sequencing (2
×
150 bp). On a e age, samples had abou
325 million eads each.
2.3. Genomic Da a and Me hyla ion P ocessing
Quali y checks and imming we e pe o med by using Fas QC [
27
]T imGalo e 0.4.5
(a w appe ool a ound Cu adap [
28
], which emo ed adap e sequences and poo -quali y
bases and eads).
The emaining high-quali y eads (a e age Ph ed sco e > 35) we e aligned o a bisul i e-
con e ed human genome (Ensembl 91 assembly, hg38) by using he Bisma k Bisul i e
Read Mappe ( 0.20.0) [
29
], which makes use o Bow ie 1.2.1.1 alignmen so wa e [
30
].
On a e age, 89% o he sequences we e aligned. In o ma ion ega ding sequencing and
alignmen da a quali y a he sample le el is epo ed in Supplemen a y Table S2.
Me hyla ion calls o e e y single C analyzed we e pe o med by he Bisma k bis-
ma k_me hyla ion_ex ac o sc ip . Fo each CpG, he be a alues (β) we e calcula ed as
β= CGme hyla ed/(CGme hyla ed + CGunme hyla ed),
whe e CGme hyla ed is he numbe o me hyla ed cy osine and (CGme hyla ed + CGun-
me hyla ed) is he sum o me hyla ed and unme hyla ed cy osine ( o al numbe o eads)
a ha posi ion. The bisul i e con e sion a e was es ima ed om cy osine (C) in non-CpG
con ex and in a e age was abo e 99% (98.8–99.7%).
CpGs we e i s selec ed based on hei co e age: CpGs no co e ed by a leas
5 uniquely mapped eads
in all samples we e disca ded o subsequen analyses, such as
hose CpG wi h high be a di e ence a iance, ending wi h 14,848,915 CpG si es.
2.4. Analyses o Di e en ially Me hyla ed Posi ion/Region
To iden i y di e en ially me hyla ed egions (DMRs), we conduc ed a pai wise analy-
sis using cus om R sc ip s, wi h be a alues as he inpu o all analyses.
Sho ly, o each il e ed CpG si e om he p e ious s ep, a pai ed Wilcoxon es was
applied o compa e me hyla ion le els. p-Values we e adjus ed o mul iple es ing by using
he Benjamini–Hochbe g (BH) algo i hm, and CpGs wi h a co ec ed p- alue less han 0.05
and a me hyla ion di e ence o a leas 0.2 be ween he wo g oups we e e ained. CpGs
loca ed a SNP posi ions wi h a popula ion global mino allele equency (MAF) g ea e
han 1% (dbSNP Build 155) we e excluded o minimize he impac o gene ic a ia ion on
me hyla ion analysis.
DMRs we e de ined by agg ega ing CpGs loca ed wi hin a maximum dis ance o
50 base pai s.
Only egions con aining h ee o mo e CpGs, wi h a leas 60% o he
CpGs in he egion mee ing he signi icance c i e ia, we e selec ed o u he analysis.
This app oach ensu ed he iden i ica ion o obus egions wi h biologically meaning ul
me hyla ion di e ences.
Cance s 2025,17, 654 5 o 19
2.5. Pa hway En ichmen Analysis
To analyze he biological meaning o ou DMRs and add an ex a laye o alida ion,
we pe o med pa hway en ichmen wi h KEGG [
31
] pa hways. As a i s s ep, an ex ensi e
mapping o he DMRs was pe o med o ind pu a i e cis- egula o y egions ela ed o
speci ic genes by using he GREAT unc ion [
32
]. A e wa ds, wi h he associa ed gene
in o ma ion, we p oceeded o pe o m pa hway en ichmen by using Clus e P o ile [
33
],
wi h a p- alue o 0.05 and limi ing he sea ch o Homo sapiens. The esul ing pa hways
we e isualized by using he R package en ichplo [34].
2.6. Copy-Numbe Al e a ion De ec ion
To e alua e la ge-scale copy-numbe al e a ions (CNAs), he ool icho CNA [
35
] was
used. Read coun s o each APL, CRC, and espec i e no mal samples we e calcula ed
wi h he HMMcopy Sui e [
36
]: each genome was di ided in o 10 kb non-o e lapping bins,
and aligned eads we e coun ed based on o e lap wi hin each bin; hen, ead coun s we e
no malized o co ec o GC con en and mappabili y biases. Cen ome es we e il e ed
based on ch omosome gap coo dina es ob ained om UCSC (Uni e si y o Cali o nia a
San a C uz) o hg38.
No mal samples we e used o c ea e a e e ence da ase . These da a we e u he used
o co ec sys ema ic biases a ising om lib a y cons uc ion, sequencing pla o m, and
DNA-speci ic a i ac s.
Copy-numbe analysis was pe o med on au osomal ch omosomes only, using icho -
CNA de aul pa ame e s.
2.7. Soma ic Mu a ion De ec ion
To o e come he bisul i e con e sion p oblem, which can cause cy osine o appea
as hymine du ing sequencing and complica e he de ec ion o ue mu a ions, a pipeline
based on EpiDi e se/snp (h ps://gi hub.com/EpiDi e se/snp (accessed on 28 Janua y
2022)) [
37
] was c ea ed. In summa y, bam iles we e i s p ep ocessed by using sam ools
(so , calmd, and index); hen, a double-masking p ocedu e, which manipula es speci ic
nucleo ides and base quali y (BQ) sco es on alignmen s, was applied.
Fo each sample pai , pileup iles we e c ea ed wi h sam ools. The soma ic unc ion
o Va Scan2 [
38
,
39
] was used o call soma ic single-nucleo ide a ian s (SNVs) and small
inse ions/dele ions (INDELs) om he compa ison be ween APLs o CRCs and NATs.
Only posi ions ha we e p esen in bo h iles and me he minimum co e age in bo h
iles we e compa ed. Only mu a ions iden i ied by he ool as soma ic we e anno a ed by
SnpE [
40
]. To summa ize, analyze, and isualize he mu a ions, he VCF ou pu om
Va Scan2 and SnpE was con e ed in o Mu a ion Anno a ion Fo ma (MAF) iles and
analyzed by using he R Bioconduc o package Ma ools [41].
2.8. CpG Island Me hyla o Pheno ype (CIMP)
To p edic he CIMP s a us o each sample, we u ilized CpGs de i ed om p omo e
egions ha exhibi ed a high s anda d de ia ion (SD > 0.2) o me hyla ion le els in umo
issues and a low me hyla ion le el (mean
β
< 0.05) in no mal issues. Consensus clus e ing
analysis based on he K-means algo i hm was pe o med by using he R package Consen-
susClus e Plus [
42
]. Gi en he limi ed numbe o a ailable samples o DNA me hyla ion
p o iling in his s udy, we classi ied samples wi h hype me hyla ion pa e ns as CIMP-H,
while all o he samples we e ca ego ized as CIMP-L/N.

Cance s 2025,17, 654 6 o 19
2.9. Mic osa elli e Ins abili y (MSI) S a us P edic ion
S anda d sho andem epea DNA sequence sea ch me hods a e no applicable o
bisul i e-con e ed da a, as he con e sion al e s he DNA sequence and p e en s he di ec
de ec ion o mic osa elli e ins abili y (MSI). To add ess his, we de e mined MSI s a us by
examining po en ial ameshi mu a ions in genes associa ed wi h he misma ch epai
(MMR) pa hway, like RNF43, PMS2, MSH6, MSH2, and MLH1. Addi ionally, we applied
he CpG bioma ke panel used in he MSIMEP me hod [
43
], o iginally de eloped o
mic oa ay DNA me hyla ion p o iling, o ou whole-genome bisul i e sequencing (WGBS)
da a. By le e aging hese es ablished bioma ke s, we adap ed he app oach o WGBS,
enabling us o es ima e MSI s a us in ou da ase e ec i ely.
2.10. P incipal Componen Analysis (PCA)
P incipal componen analysis (PCA) educes he dimensionali y o mul i a ia e da a
o wo o h ee dimensions, which can be isualized g aphically wi h minimal loss o
in o ma ion. PCA was pe o med on epigenomics da a o illus a e he di e ences be ween
APL his ologies by using he p comp unc ion in R package s a s. The R package ac oex a
was used o isualize he PCA ou pu .
3. Resul s
We ob ained 168 esh ozen samples, ully cha ac e ized clinically and his olog-
ically by using s anda dized c i e ia (desc ibed in Sec ion 2and Supplemen a y Table
S1). To ensu e obus ness and gene alizabili y, samples we e di ided in o a disco e y
da ase o 48 APL samples pai ed wi h no mal adjacen issue (NAT), a alida ion da ase
o
26 non-pai ed APL samples,
and a hi d da ase comp ising 23 pai ed CRC samples
(Table 1). The disco e y da ase was used o explo e and iden i y he APL signa u e, pe -
o ming di e en ial me hyla ion analysis and CNV/mu a ion analysis, while he alida ion
da ase was used exclusi ely o assessing ou signa u e pe o mance in he agnos ic iden-
i ica ion o APLs by using he signa u e me hod. Finally, he CRC da ase was used o
compa e and unde s and he di e ences be ween he p ecance ous s a e and he in asi e
cance s a e. The esea ch pipeline is su mised in Figu e 1.
Cance s2025,17,xFORPEERREVIEW6o 20


2.9.Mic osa elli eIns abili y(MSI)S a usP edic ion
S anda dsho  andem epea DNAsequencesea chme hodsa eno applicable o
bisul i e-con e edda a,as hecon e sional e s heDNAsequenceandp e en s hedi-
ec de ec iono mic osa elli eins abili y(MSI).Toadd ess his,wede e minedMSIs a-
usbyexaminingpo en ial ameshi mu a ionsingenesassocia edwi h hemisma ch
epai (MMR)pa hway,likeRNF43,PMS2,MSH6,MSH2,andMLH1.Addi ionally,we
applied heCpGbioma ke panelusedin heMSIMEPme hod[43],o iginallyde eloped
o mic oa ayDNAme hyla ionp o iling, oou whole-genomebisul i esequencing
(WGBS)da a.Byle e aging hesees ablishedbioma ke s,weadap ed heapp oach o
WGBS,enablingus oes ima eMSIs a usinou da ase effec i ely.
2.10.P incipalComponen Analysis(PCA)
P incipalcomponen analysis(PCA) educes hedimensionali yo mul i a ia eda a
o woo  h eedimensions,whichcanbe isualizedg aphicallywi hminimallosso in-
o ma ion.PCAwaspe o medonepigenomicsda a oillus a e hediffe encesbe ween
APLhis ologiesbyusing hep comp unc ioninRpackages a s.TheRpackage ac oex a
wasused o isualize hePCAou pu .
3.Resul s
Weob ained168 esh ozensamples, ullycha ac e izedclinicallyandhis ologi-
callybyusings anda dizedc i e ia(desc ibedinSec ion2andSupplemen a yTableS1).
Toensu e obus nessandgene alizabili y,sampleswe edi idedin oadisco e yda ase 
o 48APLsamplespai edwi hno maladjacen  issue(NAT),a alida ionda ase o 26
non-pai edAPLsamples,anda hi dda ase comp ising23pai edCRCsamples(Table
1).Thedisco e yda ase wasused oexplo eandiden i y heAPLsigna u e,pe o ming
diffe en ialme hyla ionanalysisandCNV/mu a ionanalysis,while he alida ionda ase 
wasusedexclusi ely o assessingou signa u epe o mancein heagnos iciden i ica-
iono APLsbyusing hesigna u eme hod.Finally, heCRCda ase wasused ocompa e
andunde s and hediffe encesbe ween hep ecance ouss a eand hein asi ecance 
s a e.The esea chpipelineissu misedinFigu e1.

Figu e1.Resea chpipeline.G aphical ep esen a iono pipeline ollowedin hep esen wo k.
Figu e 1. Resea ch pipeline. G aphical ep esen a ion o pipeline ollowed in he p esen wo k.
Cance s 2025,17, 654 7 o 19
3.1. DMR Analysis Shows Unique Me hyla ion Pa e ns Be ween APL Sub ypes and CRC
We conduc ed a comp ehensi e analysis o iden i y di e en ially me hyla ed egions
(DMRs) among APL sub ypes. Ou analysis employed wo key app oaches: i s , pai wise
compa isons o each APL sub ype—SSL, TA, and VA/TVA—agains hei co esponding
non-adjacen no mal issue (NAT) samples, allowing us o iden i y sub ype-speci ic me hy-
la ion changes ela i e o hei no mal issue baseline. Second, di ec compa isons among
he h ee APL sub ypes we e pe o med o unco e DMRs ha highligh me hyla ion
di e ences unique o each his ological g oup.
The esul s o hese analyses a e summa ized in Table 2, which p esen s he o al
numbe o DMRs iden i ied in each compa ison. These indings quan i y he signi i-
can me hyla ion di e ences obse ed bo h wi hin indi idual sub ypes ( ela i e o NAT)
and among he sub ypes hemsel es. In o al, 122,348 DMRs we e iden i ied, including
6263 hype me hyla ed and 116,050 hypome hyla ed egions.
To p io i ize he mos biologically ele an signals, we ocused on hype me hyla ed
DMRs unique o each APL sub ype. These egions we e selec ed based on hei absence
in NAT samples and hei dis inc p esence in a single sub ype, ensu ing he iden i i-
ca ion o obus , sub ype-speci ic signals. Figu e 2a illus a es he di e en analy ical
compa isons pe o med among he h ee APL sub ypes, wi h ed ci cles highligh ing he
unique hype me hyla ed egions ha ep esen key me hyla ion pa e ns en iched in each
his ological sub ype.
O e all, ou indings e eal ex ensi e DMRs dis inguishing APL sub ypes om one
ano he , as well as om no mal mucosa and CRC.
We ound 2003 hype me hyla ed egions ac oss he APL sub ypes which we e absen
in he no mal adjacen issues. I should be no ed ha he DMRs we e ound no o be
co ela ed wi h age (a e age Spea man co ela ion o 0.06) o sample o igin (a e age
Spea man co ela ion o 0.09).
A e analyzing he genomic dis ibu ion o DMRs, we obse ed hei widesp ead
p esence ac oss all ch omosomes (Figu e 2b). The Ci cos plo p o ides a isual ep esen a-
ion o he human genome, wi h he ou e mos ing co esponding o ch omosomes 1 o 22.
The nex wo ings depic he densi y o DMRs along he ch omosome sequences, shown as
a do plo and a densi y plo . The inne mos laye highligh s he 30 mos signi ican DMRs
iden i ied o subsequen PCA analysis, wi h each line ep esen ing a speci ic egion. Gene
names a e included when a DMR o e laps wi h a known gene.
Table 2. Summa y o DMRs in all analyses.
Compa ison DMRs Hype DMRs Hypo DMRs
SSL s. SSL NAT 6317 1271 5046
TA s. TA NAT 36,297 861 35,436
VA/TVA s. VA/TVA NAT 65,794 1807 63,987
SSL s. TA 1299 137 1162
SSL s. VA/TVA 11,955 2096 9859
TA s. VA/TVA 651 91 560
SSL NAT s. TA NAT 4
SSL NAT s. VA/TVA NAT 30
TA NAT s. VA/TVA NAT 1
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Figu e2.(a)DMRselec ion.TheUpSe plo used o isualize heanaly icalcompa isonsamong he
APLs.Toen ich hehis ologicalsub ypesignals,only hose egions ha dono o e lapwi hAPL
s.NAT esul sa econside ed( edci cles).Alliden i ied egionsand hei possiblein e sec ions
a e ep esen edbyablackdo .(b)Ci cosplo o genomic egions,displaying hedis ibu iono 
DMRsac ossallch omosomes.Theou e mos laye  ep esen s hech omosomes, ollowedbyado 
plo indica ing hegenomicposi iono eachindi idualDMR.Thenex laye isadensi yplo sum-
ma izing hequan i yo DMRsalongeachch omosome.Theinne mos laye highligh s he op30
DMRs(anno a edwi hgenenameswhenapplicable) ha signi ican lycon ibu e o hePCAanal-
ysisshowninFigu e3a.
Figu e 2. (a) DMR selec ion. The UpSe plo used o isualize he analy ical compa isons among he
APLs. To en ich he his ological sub ype signals, only hose egions ha do no o e lap wi h APL
s. NAT esul s a e conside ed ( ed ci cles). All iden i ied egions and hei possible in e sec ions
a e ep esen ed by a black do . (b) Ci cos plo o genomic egions, displaying he dis ibu ion o
DMRs ac oss all ch omosomes. The ou e mos laye ep esen s he ch omosomes, ollowed by a
do plo indica ing he genomic posi ion o each indi idual DMR. The nex laye is a densi y plo
summa izing he quan i y o DMRs along each ch omosome. The inne mos laye highligh s he
op 30 DMRs (anno a ed wi h gene names when applicable) ha signi ican ly con ibu e o he PCA
analysis shown in Figu e 3a.
Cance s 2025,17, 654 9 o 19
Cance s2025,17,xFORPEERREVIEW9o 20
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Figu e3.(a)P incipalcomponen analysis(PCA)o diffe en iallyme hyla ed egions,showing he
i s  wocomponen so  a ianceinbe a alues o SSL( ed),TA(o ange),andVA/TVA(ligh blue).
Smallsymbols ep esen indi idualsample a iabili y,whilela ge symbolsindica e hecen oid
(mean)o eachg oupinPCAspace.(b)Boxplo illus a ing hes a is icaldiffe encesin he i s 
p incipalcomponen ’scon ibu ionsamong heAPLsub ypes,wi hsigni icancede e minedby he
K uskal–Wallis es .
A e analyzing hegenomicdis ibu iono DMRs,weobse ed hei widesp ead
p esenceac ossallch omosomes(Figu e2b).TheCi cosplo p o idesa isual ep esen-
a iono  hehumangenome,wi h heou e mos  ingco esponding och omosomes1 o
22.Thenex  wo ingsdepic  hedensi yo DMRsalong hech omosomesequences,
shownasado plo andadensi yplo .Theinne mos laye highligh s he30mos 
Figu e 3. (a) P incipal componen analysis (PCA) o di e en ially me hyla ed egions, showing he
i s wo componen s o a iance in be a alues o SSL ( ed), TA (o ange), and VA/TVA (ligh blue).
Small symbols ep esen indi idual sample a iabili y, while la ge symbols indica e he cen oid
(mean) o each g oup in PCA space. (b) Boxplo illus a ing he s a is ical di e ences in he i s
p incipal componen ’s con ibu ions among he APL sub ypes, wi h signi icance de e mined by he
K uskal–Wallis es .
In o al, 803 genes associa ed wi h DMRs we e iden i ied (Supplemen a y Table S3).
Among hese, some genes, such as CDKN2A and AMER2, a e known o be epigene ically
silenced in CRC. Howe e , u he ansc ip omic analysis is needed o con i m hei
unc ional silencing, which is beyond he scope o he cu en s udy.
To u he in es iga e whe he speci ic ch omosomes exhibi a highe densi y o
DMRs, we pe o med an en ichmen analysis o DMRs pe ch omosome, no malizing
Cance s 2025,17, 654 16 o 19
The indings unde sco e he po en ial o DMRs as obus bioma ke s o ea ly de ec-
ion, o e ing oppo uni ies o imp o e diagnos ic accu acy and s a i y pa ien s based on
hei isk o p og ession o colo ec al cance (CRC). Addi ionally, he obse ed di e ences
in CNVs and soma ic mu a ions among APL sub ypes u he ein o ce he impo ance o a
mul i-omics app oach o ully cha ac e ize he molecula landscape o hese lesions.
Beyond hei diagnos ic alue, hese molecula al e a ions p esen oppo uni ies o he
de elopmen o a ge ed p e en i e s a egies. Epigene ic modi ica ions o e an a ac i e
a enue o he apeu ic in e en ion due o hei e e sible na u e. The iden i ica ion o
sub ype-speci ic al e a ions could pa e he way o pe sonalized medicine app oaches,
enabling ailo ed su eillance and ea men s a egies o indi iduals a highe isk o
CRC p og ession.
While ou s udy p o ides a comp ehensi e amewo k o unde s anding he molecu-
la biology o APLs, u he esea ch is equi ed o alida e hese indings in la ge coho s
and o explo e hei unc ional ele ance. Fu u e s udies should also in es iga e he in-
eg a ion o hese molecula signa u es in o exis ing clinical wo k lows o enhance ea ly
de ec ion and imp o e pa ien ou comes. Finally, he inco po a ion o a i icial in elligence
and machine lea ning algo i hms mus be pe o med o imp o e ea u e selec ion and
de ec ion a es.
In conclusion, his s udy con ibu es o he g owing body o knowledge on ea ly
colo ec al umo igenesis and highligh s he po en ial o mul i-omics s a egies o ans o m
he diagnosis, p e en ion, and managemen o CRC. By ad ancing ou unde s anding o
APL sub ypes, we hope o acili a e he de elopmen o no el ools and s a egies aimed a
educing he global bu den o colo ec al cance .
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/cance s17040654/s1, Figu e S1: Hea map o me hyla ion p o-
iles in all he g oups, Figu e S2: (a) Hea map o CIMP s a us. (b) Hea map o MSI s a us,
Table S1: Desc ip ion
o he samples included in he s udy, Table S2: Da a quali y me ics pe sample,
Table S3: Lis o genes wi h a leas one associa ed DMR, Table S4: Comp ehensi e lis o soma ic
mu a ions iden i ied in he s udy.
Au ho Con ibu ions: Concep ualiza ion, F.M.M. and A.B.; me hodology, F.M.M.; so wa e, F.M.M.;
alida ion, F.M.M.; o mal analysis F.M.M.; esou ces, P.C.-N. and K.K.; da a cu a ion, F.M.M.;
w i ing—o iginal d a p epa a ion, J.C.H.-A.; w i ing— e iew and edi ing, J.C.H.-A., F.M.M.,
P.C.-N.,
A.P.-L., M.H.A.R., and K.K.; isualiza ion, F.M.M.; supe ision, P.C.-N., K.K., M.H.A.R., and A.P.-L.;
p ojec adminis a ion, P.C.-N. and K.K.; unding acquisi ion, P.C.-N. and K.K. All au ho s ha e ead
and ag eed o he published e sion o he manusc ip .
Funding: Thiswo kwassuppo edbyg an PID2021-122952OB-I00 undedbyAEI 10.13039/501100011033
and by he ERDF—A way o making Eu ope; he Ne wo king Biomedical Resea ch Cen e in he
subjec a ea o Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN); he CERCA P o-
g amme/Gene ali a de Ca alunya (B2SLab is ce i ied as 2021 SGR 01052); and p i a e unding by
Uni e salDx S.A.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : The da a p esen ed in his s udy a e a ailable on a easonable eques
om he co esponding au ho .
Acknowledgmen s: We would like o exp ess ou since e g a i ude o Ka h yn Lang o he aluable
assis ance wi h e iewing and e ining he manusc ip and o p o iding insigh ul eedback ha
signi ican ly enhanced he quali y o his wo k.

Cance s 2025,17, 654 17 o 19
Con lic s o In e es : F.M.M., J.C.H.-A., and P.C.-N. a e employees o Uni e sal Diagnos ics S.A.,
K.K., and A.B. a e employees o Uni e salDx d.o.o. The emaining au ho s decla e ha he esea ch
was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as
a po en ial con lic o in e es . The unde s had no ole in he design o he s udy; in he collec ion,
analyses, o in e p e a ion o da a; in he w i ing o he manusc ip ; o in he decision o publish
he esul s.
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